This is a single central aim project to investigate the role of autologous endothelial cells (ECs) in regulating the generation of definitive hematopoietic stem/progenitor cells (HSPCs) from human pluripotent stem cells (hPSCs). The transplantation of hematopoietic stem cells (HSCs) allows for the cure of patients with hematological disorders. Because of limited availability of HSCs from donors, derivation of HSCs from hPSCs provides an alternative source of HSCs for bone marrow transplantation. However, generation of hPSCs-derived HSCs that are capable of long-term engraftment and differentiation to multilineage hematopoietic cells remain significant challenges, which limits progress of the clinical applications of stem cell therapy and gene therapy using patient-specific induced pluripotent stem cells (hiPSCs). The difficulty to efficiently generate HSCs from hPSCs is possibly due to a restricted temporal window and demanding niche cues for HSC emergence and development from hPSCs. During embryonic development, hemogenic ECs, a specified subset of ECs in vascular endothelium of aorta-gonad-mesonephros (AGM), give rise to multipotent HSCs. We hypothesized that during hPSC differentiation, autologous ECs provide vascular niche to promote the generation of definitive HSCs from hemogenic ECs in multiple stages, including hemogenic EC-to-hematopoietic transition, pre-HSC maturation to HSCs, and HSC expansion. We have developed a novel reporter system to monitor definitive HSC emergence from hemogenic ECs by utilizing Runx1 enhancer and minimal promotor of Wiskott-Aldrich Syndrome (WAS) gene. Using this reporter system and cell immunophenotypes, we will test our hypothesis, and investigate the role of extracellular ATP/adenosine from vascular niche in regulating the generation of definitive hematopoietic cells from hemogenic ECs. CD73, an ecto-5'-nucleotidase that convert AMP to adenosine at cell surface, plays a key role in extracellular ATP metabolism and the generation of adenosine. Adenosine-receptor signaling promotes definitive HSPC generation in AGM. However, CD73 is absent in hemogenic ECs. We will use CD73-deficient hiPSCs and pharmacological regents to investigate how extracellular ATP/adenosine regulates definitive HSPC generation. To evaluate their functionalities of repopulation and differentiation in vivo, we will characterize hematopoietic stem and progenitor cells derived from hemogenic ECs by a human-to-mouse xenogeneic transplantation model, which represents powerful tools and essential preclinical platform for assessing human HSC function. Our investigation will provide new insight into the human HSPC generation regulation mechanism, accelerate the establishment of a novel strategy to generate hematopoietic stem and progenitor cells, and lead to a preclinical study for cell-based therapy for hematological disorders.